Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 30, 2015). doi:10.1152/ajplung.00286.2014 Manuscript for the American Journal of Physiology - Lung Cellular and Molecular Physiology
1
Pericytes contribute to airway remodeling in a mouse model of chronic allergic
2
asthma
3 4
Jill R. Johnson1, 2, *, Erika Folestad1, Jessica E. Rowley2, Elisa M. Noll2, Simone A.
5
Walker2, Clare M. Lloyd2, Sara M. Rankin2, Kristian Pietras1, 3, Ulf Eriksson1 and
6
Jonas Fuxe1
7 8
1
9
Vascular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Department of Medical Biochemistry and Biophysics, Matrix Division, Division of
10
2
11
Building, Imperial College London, London SW7 2AZ, United Kingdom
12
3
Leukocyte Biology Section, National Heart and Lung Institute, Sir Alexander Fleming
Lund University, Department of Laboratory Medicine Lund, SE-223 81 Lund, Sweden
13 14
*Corresponding author: Jill Johnson, PhD, Leukocyte Biology Section, National Heart
15
and Lung Institute, Sir Alexander Fleming Building, Imperial College London, London
16
SW7
17
[email protected]
2AZ,
United
Kingdom.
Phone:
+44
(0)
791-448-5692.
18
Copyright © 2015 by the American Physiological Society.
Email:
Johnson et al.
Pericytes in airway remodeling
19
Abstract
20
Myofibroblast accumulation, subepithelial fibrosis and vascular remodeling are
21
complicating features of chronic asthma, but the mechanisms are not clear. Platelet-
22
derived growth factors (PDGFs) regulate the fate and function of various mesenchymal
23
cells and have been implicated as mediators of lung fibrosis. However, it is not known
24
whether PDGF-BB signaling via PDGFR, which is critical for the recruitment of
25
pericytes to blood vessels, plays a role in airway remodeling in chronic asthma.
26
In the present study, we used a selective PDGFRβ inhibitor (CP-673,451) to
27
investigate the role of PDGFR signaling in the development of airway remodeling and
28
lung dysfunction in an established mouse model of house dust mite (HDM) induced
29
chronic allergic asthma.
30
Unexpectedly, we found that pharmacological inhibition of PDGFRβ signaling in
31
the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and
32
airway smooth muscle thickening. Further studies revealed that the inflammatory
33
response to aeroallergen challenge in mice was associated with decreased PDGF-BB
34
expression and the loss of pericytes from the airway microvasculature. In parallel, cells
35
positive for pericyte markers accumulated in the subepithelial region of chronically
36
inflamed airways. This process was exacerbated in animals treated with CP-673,451.
37
The results indicate that perturbed PDGF-BB/PDGFR signaling and pericyte
38
accumulation in the airway wall may contribute to airway remodeling in chronic allergic
39
asthma.
40
Keywords: asthma, house dust mite, airway hyperresponsiveness, pericyte, remodeling
2
Johnson et al.
41
Pericytes in airway remodeling
Introduction
42
Asthma is a heterogenic disease that currently affects 300 million people
43
worldwide (34). Allergic asthma is defined as an allergen-induced, immune-driven
44
chronic lung disease manifested by recurrent episodes of airway inflammation
45
composed of infiltrating eosinophils, mast cells, macrophages, neutrophils and
46
lymphocytes (1). Airway remodeling is thought to be facilitated by this chronic
47
inflammatory process (5). The structural changes associated with airway remodeling
48
comprise goblet cell hyperplasia, collagen deposition and increased airway smooth
49
muscle (ASM) mass, all of which contribute to the clinical manifestation of lung
50
dysfunction (1). Inflammatory cytokines and the pro-fibrogenic growth factor
51
transforming growth factor-β (TGF-β) are believed to play significant roles in driving
52
these structural changes. However, the signaling mechanisms and the cellular sources
53
from which the increased mesenchymal cell population of the airway wall is derived
54
have not been fully described. Some studies have demonstrated that bone marrow-
55
derived fibrocytes contribute to airway wall remodeling (30, 31), while others have
56
suggested a minimal role for these cells (24). Additionally, the differentiation of
57
mesenchymal cells from airway epithelial cells via epithelial-to-mesenchymal transition
58
has been shown to be a mechanism of remodeling in a mouse model of severe allergic
59
airways disease (20).
60
The platelet-derived growth factors (PDGFs) are mitogens for a number of
61
mesenchymal cell types, including fibroblasts and smooth muscle cells (9). The
62
receptors of the PDGFs, PDGF receptor alpha (PDGFRα) and PDGF receptor beta
63
(PDGFRβ) are tyrosine kinases, and are thus amenable to pharmacological
3
Johnson et al.
Pericytes in airway remodeling
64
intervention. However, little is known regarding the expression of the PDGFs in asthma,
65
as the limited studies available in the literature have documented few differences in
66
PDGF or PDGF receptor expression in human asthmatics compared to healthy controls
67
(3, 6, 15, 25). Specifically focusing on PDGF-BB and its cognate receptor PDGFRβ in
68
the context of allergic airway disease, stimulation of ASM cells with PDGF-BB in vitro
69
has been shown to act in concert with TGF-β to stimulate cell migration (18). Moreover,
70
a recent study using an adenovirus vector to overexpress PDGF-BB in the airway
71
epithelium in an OVA-driven mouse model of asthma was shown to induce ASM cell
72
proliferation and enhance airway hyperresponsiveness (14). PDGF receptors are also
73
expressed on vascular mural cells, a heterogeneous population of mesenchymal cells
74
that line the outer surface of microvessels and are therefore abundant in the lung (2).
75
Pericytes, the population of mural cells covering capillaries, express desmin and NG2
76
but are negative for α-smooth muscle actin (α-SMA), while mural cells covering
77
arterioles and venules express desmin and α-SMA and are termed vascular smooth
78
muscle (VSM) cells (2). Mural cells are recruited to and retained on blood vessels
79
through PDGF-BB/PDGFRβ interactions (reviewed in (2)). Impaired pericyte coverage
80
of blood vessels is seen in PDGF-BB-deficient mice and in diseases like cancer, and is
81
associated with vascular leakage and edema (2, 4, 32).
82
In light of these findings, and since tyrosine kinase inhibitors such as masitinib
83
are currently being investigated as asthma therapies (16), we elected to investigate the
84
impact of PDGFRβ inhibition on airway and VSM cells/pericytes in a mouse model of
85
chronic aeroallergen exposure driven by exposure to house dust mite (HDM) extract via
86
the respiratory mucosa. HDM exposure is strongly associated with human asthma and
4
Johnson et al.
Pericytes in airway remodeling
87
is one of the most ubiquitous respiratory allergens worldwide. In mice, chronic
88
respiratory HDM exposure leads to Th2-polarized airway inflammation, remodeling of
89
the airway wall and bronchial hyperreactivity, and thus recapitulates many of the
90
features of clinical asthma (21). Using this paradigm, we investigated the role of
91
PDGFRβ signaling and the downstream effects of inhibiting this receptor during chronic
92
HDM exposure on airway remodeling and lung dysfunction.
93 94
Materials and methods
95
Animal handling
96
Female C57Bl/6 mice were bred in-house at the Karolinska Institutet animal facility at
97
the Department of Mikrobiologi, Tumör- och Cellbiologi (MTC) or purchased from Harlan
98
Laboratories (Wyton, UK) and housed at the central animal facility at Imperial College
99
London. Transgenic mice used for pericyte lineage tracing studies (Tg(Cspg4-
100
DsRed.T1)1Akik/J) were obtained from the Jackson Laboratories (Bar Harbor, ME); the
101
phenotype of these mice was determined by direct fluorescent imaging of the DsRed
102
fluorescent signal in ear biopsies. Animals were initiated into experiments at 8-12 weeks
103
of age. Mice were housed under specific pathogen-free conditions following a 12-h light-
104
dark cycle and were provided food and water ad libitum. All experiments described in
105
this study were approved by the Research Ethics Committees at the Karolinska Institute
106
and at Imperial College London and were performed in accordance with the UK Home
107
Office and Imperial College London guidelines on animal welfare.
108 109
Antigen and drug administration 5
Johnson et al.
Pericytes in airway remodeling
110
Mice were challenged with purified Dermatophagoides pteronyssinus extract (HDM)
111
(Greer Laboratories, Lenoir, NC, USA) 5 days per week for 3 or 5 consecutive weeks.
112
HDM was re-suspended in sterile phosphate buffered saline (PBS) (2.5 mg of
113
protein/ml); 10 μl of HDM were administered intranasally to isoflurane-anesthetized
114
mice (Sigma-Aldrich, Gillingham, UK) (21). Control mice were subjected to the same
115
exposure protocol but received PBS (10 μl). In each group, a subset of animals received
116
a selective PDGFRβ inhibitor (CP-673,451 (Pfizer Inc., New York, NY, USA)) by oral
117
gavage (33 mg/kg in polyethyleneglycol 400 (PEG-400; Sigma-Aldrich) daily for a period
118
of 5 consecutive weeks. Other animals received the drug vehicle (PEG-400). 1-(2-(5-(2-
119
methoxy-ethoxy)-benzoimidazol-1-yl)-quinolin-8-yl)-piperdin-4-ylamine
120
has 10-fold higher affinity for PDGFR versus PDGFR, and greater than 450-fold
121
higher affinity for PDGFR versus c-kit, and was prepared according to the procedures
122
described previously (27).
(CP-673,451),
123 124
Lung function measurements
125
After 5 weeks of allergen and drug administration, mice were subjected to lung function
126
measurements (8). Mice were injected intraperitoneally with sodium pentobarbital (50
127
mg/kg) (Sigma-Aldrich), followed by an intramuscular injection of ketamine (90 mg/kg)
128
(Sigma-Aldrich) (29). Mice were tracheostomized using a 19-gauge blunt-ended needle.
129
Measurements of dynamic airway hyperresponsiveness (AHR) and elastance were
130
acquired using the flexiVent system (Scireq, Montréal, Canada). Mice were ventilated,
131
and airway resistance and elastance were calculated as previously described (29). A 4-
132
6 µm nebulizer was used, and the duration of nebulization was 10 seconds per dose. A
6
Johnson et al.
Pericytes in airway remodeling
133
50% duty cycle was used during nebulization. Fluctuations in AHR and elastance were
134
determined by the responses of the total respiratory system to increasing
135
concentrations (10-300 mg/ml) of methacholine (MCh) (Sigma-Aldrich) delivered into
136
the inspiratory line of the flexiVent ventilator. The PaO waveform was monitored
137
throughout flexiVent data acquisition and was found to be similar during measurements
138
as when the initial calibration was performed before running each subject.
139
Measurements were not taken when there were obvious signs of respiratory effort.
140
Following flexiVent data acquisition, the thoracic cavity of each mouse was opened to
141
confirm that cardiac arrest had not occurred.
142 143
Collection of specimens
144
Mice were humanely sacrificed and the lungs were dissected. In some experiments,
145
bronchoalveolar lavage (BAL) was collected (0.45 ml of PBS). BAL cells were counted
146
using a hemocytometer, centrifuged at 1200 rpm for 5 minutes, then mounted on glass
147
slides using a Cytospin (Thermo Scientific, Hemel Hempstead, UK) and stained with
148
hematoxylin and eosin for differential cell counts. BAL supernatants were stored at -
149
20ºC, then submitted to ELISA to determine mouse PDGF-BB expression according to
150
the manufacturer’s instructions (R&D Systems, Abingdon, UK). In other experiments,
151
mice were perfused through the left ventricle with 1% paraformaldehyde, and the
152
trachea, extrapulmonary bronchi and lung were dissected out. The tracheas,
153
extrapulmonary
154
paraformaldehyde for 30 min, then transferred to PBS. The lung was embedded in
bronchi
and
lungs
were
isolated
and
preserved
in
1%
7
Johnson et al.
Pericytes in airway remodeling
155
OCT, stored at -80°C and cut into 10 μm thick sections for staining. Fresh lung samples
156
were snap-frozen for qPCR and immunoblotting experiments.
157 158
Immunoblotting
159
Frozen lung tissue specimens were homogenized in 400 μl of lysis buffer, which was
160
composed of T-PER Tissue Protein Extraction Reagent (Rockford, IL) supplemented
161
with protease inhibitors (Complete Mini tablets from Roche Diagnostics Scandinavia,
162
Bromma, Sweden), and homogenized with a homogenizer (Percell, Stockholm,
163
Sweden). Protein samples were separated by SDS-PAGE on 4-12% polyacrylamide
164
gels and blotted onto nitrocellulose membranes. The membranes were incubated in
165
blocking buffer (5% skim milk) in Tris-buffered saline containing 0.5% Tween for 1 hour
166
at room temperature and probed with a polyclonal rabbit-antihuman PDGF-BB antibody
167
(Aviva Systems Biology, San Diego, CA) diluted 1:1000 in blocking buffer. After
168
washing, the membranes were incubated with a HRP-conjugated secondary anti-rabbit
169
antibody (GE Healthcare, Stockholm, Sweden) diluted 1:5000 in blocking buffer, for 1
170
hour at RT. The membranes were developed using ECL+ detection reagent
171
(Amersham, GE Healthcare). To ascertain equal loading of protein, the membranes
172
were stripped and re-probed with a rabbit-anti-mouse antibody against Akt (Cell
173
Signaling/BioNordika Sweden, Stockholm, Sweden) diluted 1:1000 in blocking buffer.
174 175
Immunofluorescent staining
176
Lung sections from C57Bl6 and DsRed-NG2 mice were warmed to room temperature
177
and non-specific binding was blocked by incubating in 5% normal goat serum (NGS),
8
Johnson et al.
Pericytes in airway remodeling
178
0.3% Triton-X 100 and 0.1% bovine serum albumin (BSA) (all purchased from Sigma-
179
Aldrich) in PBS for 1 h at RT. Sections were stained using the appropriate primary (O/N)
180
and secondary (1 h) antibodies (Table 1). After washing twice in PBS, sections were
181
mounted using a glycerol-based mounting medium containing DAPI (Vector
182
Laboratories, Peterborough, UK) to visualize DNA. For polyclonal antibodies, negative
183
reagent controls were carried out.
184
For
tracheobronchal
whole
mount
immunostaining,
the
trachea
and
185
extrapulmonary bronchi were dissected and cleaned of connective tissue and mounted
186
on KwikgardTM-coated (WPI, Sarasota, FL, USA) 6-well culture plates. Non-specific
187
binding was blocked by incubating trachea sections in 5% NGS, 0.3% Triton-X 100 and
188
0.2% BSA in PBS O/N. Left and right trachea sections were stained using the
189
appropriate primary and secondary antibodies O/N at RT (Table 1). Sections were
190
washed thrice in PBS and mounted using a glycerol-based mounting medium containing
191
DAPI (Vector Laboratories). For all antibodies, negative reagent controls were carried
192
out with no staining observed.
193 194
Light microscopy
195
Quantification of αSMA and NG2 positive cells in the trachea and extrapulmonary
196
bronchi was performed using a Leica DM2500 fluorescence microscope (Leica
197
Microsystems, Milton Keynes, UK). Counts were normalized to the length of each
198
tracheobronchal whole mount (including the extrapulmonary bronchi at the distal end of
199
the preparation). For image acquisition from immunostained tracheobronchal whole
200
mounts and lung sections, 1024 x 1024 pixel RGB-color images were obtained using a
9
Johnson et al.
Pericytes in airway remodeling
201
Zeiss LSM 510 inverted confocal microscope with argon, helium-neon and UV lasers
202
(Carl Zeiss AG, Göttingen, Germany) using an EC Plan-Neofluar 40x/1.30 oil objective
203
with the DIC phase contrast technique (10). Using the optimized pinhole size, 0.9 μm
204
thick optical sections were sequentially collected with a step size of 1 μm (lung sections)
205
or 1.5 μm (tracheas). Image analysis was performed using Zeiss AIM 3.2.2 software
206
and Zeiss LSM Image software (Carl Zeiss AG). Three-dimensional visualizations
207
(Videos 1-4) of confocal images were prepared using Volocity software (Perkin-Elmer,
208
Waltham, MA). Morphometric analysis of ASM thickness was performed by a blinded
209
observer on αSMA-stained lung sections and analyzed using ImageJ software (NIH,
210
USA). Morphometric analysis of the number of NG2+ cells in the airway subepithelial
211
region was conducted by a blinded observer on lung sections stained for NG2, SMA
212
and CD31 (to prevent enumeration of pericytes associated with the microvasculature).
213
Five representative images were taken of the large airways (bronchi and bronchioles)
214
from each animal (n=5-10 per group from two independent experiments). Images were
215
taken on a Zeiss LSM 510 inverted confocal microscope and processed using ImageJ.
216
NG2+ cells in the airway subepithelial region were enumerated and counts were
217
normalized to mm of basement membrane.
218 219
Quantitative PCR (qPCR)
220
Whole tissue RNA from lung was isolated with TRIzol reagent (Invitrogen/Life
221
Technologies, Stockholm, Sweden) and QIAGEN RNeasy (Qiagen, Sollentuna,
222
Sweden) according to the manufacturers’ instructions. Total RNA (1 μg) was reverse
223
transcribed according to the manufacturer’s instructions (iScript cDNA synthesis kit, Bio-
10
Johnson et al.
Pericytes in airway remodeling
224
Rad). qPCR was performed using Platinum SYBR green SuperMix (Invitrogen) and
225
25 ng cDNA per reaction. Primers used were: Pdgfb QT00266910, Pdgfrb QT00113148,
226
L19 QT01779218. Ct values for the three genes analyzed were: 19-20 (PDGF-BB); 24-
227
26 (PDGFRβ); 17-18 (L19). Expression levels were normalized to the expression of
228
L19. Melt curves were assessed following the completion of RT-PCR to ensure that the
229
fluorescent signal resulted from a single PCR product.
230 231
Data analysis
232
Data were analyzed with GraphPad Prism Software version 6 (GraphPad Software, San
233
Diego, CA, USA). Statistical analysis was performed by Student’s t-test or one-way
234
analysis of variance (ANOVA) with a subsequent Tukey post-hoc test where
235
appropriate. Differences were considered statistically significant when p
1
Pericytes contribute to airway remodeling in a mouse model of chronic allergic
2
asthma
3 4
Jill R. Johnson1, 2, *, Erika Folestad1, Jessica E. Rowley2, Elisa M. Noll2, Simone A.
5
Walker2, Clare M. Lloyd2, Sara M. Rankin2, Kristian Pietras1, 3, Ulf Eriksson1 and
6
Jonas Fuxe1
7 8
1
9
Vascular Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Department of Medical Biochemistry and Biophysics, Matrix Division, Division of
10
2
11
Building, Imperial College London, London SW7 2AZ, United Kingdom
12
3
Leukocyte Biology Section, National Heart and Lung Institute, Sir Alexander Fleming
Lund University, Department of Laboratory Medicine Lund, SE-223 81 Lund, Sweden
13 14
*Corresponding author: Jill Johnson, PhD, Leukocyte Biology Section, National Heart
15
and Lung Institute, Sir Alexander Fleming Building, Imperial College London, London
16
SW7
17
[email protected]
2AZ,
United
Kingdom.
Phone:
+44
(0)
791-448-5692.
18
Copyright © 2015 by the American Physiological Society.
Email:
Johnson et al.
Pericytes in airway remodeling
19
Abstract
20
Myofibroblast accumulation, subepithelial fibrosis and vascular remodeling are
21
complicating features of chronic asthma, but the mechanisms are not clear. Platelet-
22
derived growth factors (PDGFs) regulate the fate and function of various mesenchymal
23
cells and have been implicated as mediators of lung fibrosis. However, it is not known
24
whether PDGF-BB signaling via PDGFR, which is critical for the recruitment of
25
pericytes to blood vessels, plays a role in airway remodeling in chronic asthma.
26
In the present study, we used a selective PDGFRβ inhibitor (CP-673,451) to
27
investigate the role of PDGFR signaling in the development of airway remodeling and
28
lung dysfunction in an established mouse model of house dust mite (HDM) induced
29
chronic allergic asthma.
30
Unexpectedly, we found that pharmacological inhibition of PDGFRβ signaling in
31
the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and
32
airway smooth muscle thickening. Further studies revealed that the inflammatory
33
response to aeroallergen challenge in mice was associated with decreased PDGF-BB
34
expression and the loss of pericytes from the airway microvasculature. In parallel, cells
35
positive for pericyte markers accumulated in the subepithelial region of chronically
36
inflamed airways. This process was exacerbated in animals treated with CP-673,451.
37
The results indicate that perturbed PDGF-BB/PDGFR signaling and pericyte
38
accumulation in the airway wall may contribute to airway remodeling in chronic allergic
39
asthma.
40
Keywords: asthma, house dust mite, airway hyperresponsiveness, pericyte, remodeling
2
Johnson et al.
41
Pericytes in airway remodeling
Introduction
42
Asthma is a heterogenic disease that currently affects 300 million people
43
worldwide (34). Allergic asthma is defined as an allergen-induced, immune-driven
44
chronic lung disease manifested by recurrent episodes of airway inflammation
45
composed of infiltrating eosinophils, mast cells, macrophages, neutrophils and
46
lymphocytes (1). Airway remodeling is thought to be facilitated by this chronic
47
inflammatory process (5). The structural changes associated with airway remodeling
48
comprise goblet cell hyperplasia, collagen deposition and increased airway smooth
49
muscle (ASM) mass, all of which contribute to the clinical manifestation of lung
50
dysfunction (1). Inflammatory cytokines and the pro-fibrogenic growth factor
51
transforming growth factor-β (TGF-β) are believed to play significant roles in driving
52
these structural changes. However, the signaling mechanisms and the cellular sources
53
from which the increased mesenchymal cell population of the airway wall is derived
54
have not been fully described. Some studies have demonstrated that bone marrow-
55
derived fibrocytes contribute to airway wall remodeling (30, 31), while others have
56
suggested a minimal role for these cells (24). Additionally, the differentiation of
57
mesenchymal cells from airway epithelial cells via epithelial-to-mesenchymal transition
58
has been shown to be a mechanism of remodeling in a mouse model of severe allergic
59
airways disease (20).
60
The platelet-derived growth factors (PDGFs) are mitogens for a number of
61
mesenchymal cell types, including fibroblasts and smooth muscle cells (9). The
62
receptors of the PDGFs, PDGF receptor alpha (PDGFRα) and PDGF receptor beta
63
(PDGFRβ) are tyrosine kinases, and are thus amenable to pharmacological
3
Johnson et al.
Pericytes in airway remodeling
64
intervention. However, little is known regarding the expression of the PDGFs in asthma,
65
as the limited studies available in the literature have documented few differences in
66
PDGF or PDGF receptor expression in human asthmatics compared to healthy controls
67
(3, 6, 15, 25). Specifically focusing on PDGF-BB and its cognate receptor PDGFRβ in
68
the context of allergic airway disease, stimulation of ASM cells with PDGF-BB in vitro
69
has been shown to act in concert with TGF-β to stimulate cell migration (18). Moreover,
70
a recent study using an adenovirus vector to overexpress PDGF-BB in the airway
71
epithelium in an OVA-driven mouse model of asthma was shown to induce ASM cell
72
proliferation and enhance airway hyperresponsiveness (14). PDGF receptors are also
73
expressed on vascular mural cells, a heterogeneous population of mesenchymal cells
74
that line the outer surface of microvessels and are therefore abundant in the lung (2).
75
Pericytes, the population of mural cells covering capillaries, express desmin and NG2
76
but are negative for α-smooth muscle actin (α-SMA), while mural cells covering
77
arterioles and venules express desmin and α-SMA and are termed vascular smooth
78
muscle (VSM) cells (2). Mural cells are recruited to and retained on blood vessels
79
through PDGF-BB/PDGFRβ interactions (reviewed in (2)). Impaired pericyte coverage
80
of blood vessels is seen in PDGF-BB-deficient mice and in diseases like cancer, and is
81
associated with vascular leakage and edema (2, 4, 32).
82
In light of these findings, and since tyrosine kinase inhibitors such as masitinib
83
are currently being investigated as asthma therapies (16), we elected to investigate the
84
impact of PDGFRβ inhibition on airway and VSM cells/pericytes in a mouse model of
85
chronic aeroallergen exposure driven by exposure to house dust mite (HDM) extract via
86
the respiratory mucosa. HDM exposure is strongly associated with human asthma and
4
Johnson et al.
Pericytes in airway remodeling
87
is one of the most ubiquitous respiratory allergens worldwide. In mice, chronic
88
respiratory HDM exposure leads to Th2-polarized airway inflammation, remodeling of
89
the airway wall and bronchial hyperreactivity, and thus recapitulates many of the
90
features of clinical asthma (21). Using this paradigm, we investigated the role of
91
PDGFRβ signaling and the downstream effects of inhibiting this receptor during chronic
92
HDM exposure on airway remodeling and lung dysfunction.
93 94
Materials and methods
95
Animal handling
96
Female C57Bl/6 mice were bred in-house at the Karolinska Institutet animal facility at
97
the Department of Mikrobiologi, Tumör- och Cellbiologi (MTC) or purchased from Harlan
98
Laboratories (Wyton, UK) and housed at the central animal facility at Imperial College
99
London. Transgenic mice used for pericyte lineage tracing studies (Tg(Cspg4-
100
DsRed.T1)1Akik/J) were obtained from the Jackson Laboratories (Bar Harbor, ME); the
101
phenotype of these mice was determined by direct fluorescent imaging of the DsRed
102
fluorescent signal in ear biopsies. Animals were initiated into experiments at 8-12 weeks
103
of age. Mice were housed under specific pathogen-free conditions following a 12-h light-
104
dark cycle and were provided food and water ad libitum. All experiments described in
105
this study were approved by the Research Ethics Committees at the Karolinska Institute
106
and at Imperial College London and were performed in accordance with the UK Home
107
Office and Imperial College London guidelines on animal welfare.
108 109
Antigen and drug administration 5
Johnson et al.
Pericytes in airway remodeling
110
Mice were challenged with purified Dermatophagoides pteronyssinus extract (HDM)
111
(Greer Laboratories, Lenoir, NC, USA) 5 days per week for 3 or 5 consecutive weeks.
112
HDM was re-suspended in sterile phosphate buffered saline (PBS) (2.5 mg of
113
protein/ml); 10 μl of HDM were administered intranasally to isoflurane-anesthetized
114
mice (Sigma-Aldrich, Gillingham, UK) (21). Control mice were subjected to the same
115
exposure protocol but received PBS (10 μl). In each group, a subset of animals received
116
a selective PDGFRβ inhibitor (CP-673,451 (Pfizer Inc., New York, NY, USA)) by oral
117
gavage (33 mg/kg in polyethyleneglycol 400 (PEG-400; Sigma-Aldrich) daily for a period
118
of 5 consecutive weeks. Other animals received the drug vehicle (PEG-400). 1-(2-(5-(2-
119
methoxy-ethoxy)-benzoimidazol-1-yl)-quinolin-8-yl)-piperdin-4-ylamine
120
has 10-fold higher affinity for PDGFR versus PDGFR, and greater than 450-fold
121
higher affinity for PDGFR versus c-kit, and was prepared according to the procedures
122
described previously (27).
(CP-673,451),
123 124
Lung function measurements
125
After 5 weeks of allergen and drug administration, mice were subjected to lung function
126
measurements (8). Mice were injected intraperitoneally with sodium pentobarbital (50
127
mg/kg) (Sigma-Aldrich), followed by an intramuscular injection of ketamine (90 mg/kg)
128
(Sigma-Aldrich) (29). Mice were tracheostomized using a 19-gauge blunt-ended needle.
129
Measurements of dynamic airway hyperresponsiveness (AHR) and elastance were
130
acquired using the flexiVent system (Scireq, Montréal, Canada). Mice were ventilated,
131
and airway resistance and elastance were calculated as previously described (29). A 4-
132
6 µm nebulizer was used, and the duration of nebulization was 10 seconds per dose. A
6
Johnson et al.
Pericytes in airway remodeling
133
50% duty cycle was used during nebulization. Fluctuations in AHR and elastance were
134
determined by the responses of the total respiratory system to increasing
135
concentrations (10-300 mg/ml) of methacholine (MCh) (Sigma-Aldrich) delivered into
136
the inspiratory line of the flexiVent ventilator. The PaO waveform was monitored
137
throughout flexiVent data acquisition and was found to be similar during measurements
138
as when the initial calibration was performed before running each subject.
139
Measurements were not taken when there were obvious signs of respiratory effort.
140
Following flexiVent data acquisition, the thoracic cavity of each mouse was opened to
141
confirm that cardiac arrest had not occurred.
142 143
Collection of specimens
144
Mice were humanely sacrificed and the lungs were dissected. In some experiments,
145
bronchoalveolar lavage (BAL) was collected (0.45 ml of PBS). BAL cells were counted
146
using a hemocytometer, centrifuged at 1200 rpm for 5 minutes, then mounted on glass
147
slides using a Cytospin (Thermo Scientific, Hemel Hempstead, UK) and stained with
148
hematoxylin and eosin for differential cell counts. BAL supernatants were stored at -
149
20ºC, then submitted to ELISA to determine mouse PDGF-BB expression according to
150
the manufacturer’s instructions (R&D Systems, Abingdon, UK). In other experiments,
151
mice were perfused through the left ventricle with 1% paraformaldehyde, and the
152
trachea, extrapulmonary bronchi and lung were dissected out. The tracheas,
153
extrapulmonary
154
paraformaldehyde for 30 min, then transferred to PBS. The lung was embedded in
bronchi
and
lungs
were
isolated
and
preserved
in
1%
7
Johnson et al.
Pericytes in airway remodeling
155
OCT, stored at -80°C and cut into 10 μm thick sections for staining. Fresh lung samples
156
were snap-frozen for qPCR and immunoblotting experiments.
157 158
Immunoblotting
159
Frozen lung tissue specimens were homogenized in 400 μl of lysis buffer, which was
160
composed of T-PER Tissue Protein Extraction Reagent (Rockford, IL) supplemented
161
with protease inhibitors (Complete Mini tablets from Roche Diagnostics Scandinavia,
162
Bromma, Sweden), and homogenized with a homogenizer (Percell, Stockholm,
163
Sweden). Protein samples were separated by SDS-PAGE on 4-12% polyacrylamide
164
gels and blotted onto nitrocellulose membranes. The membranes were incubated in
165
blocking buffer (5% skim milk) in Tris-buffered saline containing 0.5% Tween for 1 hour
166
at room temperature and probed with a polyclonal rabbit-antihuman PDGF-BB antibody
167
(Aviva Systems Biology, San Diego, CA) diluted 1:1000 in blocking buffer. After
168
washing, the membranes were incubated with a HRP-conjugated secondary anti-rabbit
169
antibody (GE Healthcare, Stockholm, Sweden) diluted 1:5000 in blocking buffer, for 1
170
hour at RT. The membranes were developed using ECL+ detection reagent
171
(Amersham, GE Healthcare). To ascertain equal loading of protein, the membranes
172
were stripped and re-probed with a rabbit-anti-mouse antibody against Akt (Cell
173
Signaling/BioNordika Sweden, Stockholm, Sweden) diluted 1:1000 in blocking buffer.
174 175
Immunofluorescent staining
176
Lung sections from C57Bl6 and DsRed-NG2 mice were warmed to room temperature
177
and non-specific binding was blocked by incubating in 5% normal goat serum (NGS),
8
Johnson et al.
Pericytes in airway remodeling
178
0.3% Triton-X 100 and 0.1% bovine serum albumin (BSA) (all purchased from Sigma-
179
Aldrich) in PBS for 1 h at RT. Sections were stained using the appropriate primary (O/N)
180
and secondary (1 h) antibodies (Table 1). After washing twice in PBS, sections were
181
mounted using a glycerol-based mounting medium containing DAPI (Vector
182
Laboratories, Peterborough, UK) to visualize DNA. For polyclonal antibodies, negative
183
reagent controls were carried out.
184
For
tracheobronchal
whole
mount
immunostaining,
the
trachea
and
185
extrapulmonary bronchi were dissected and cleaned of connective tissue and mounted
186
on KwikgardTM-coated (WPI, Sarasota, FL, USA) 6-well culture plates. Non-specific
187
binding was blocked by incubating trachea sections in 5% NGS, 0.3% Triton-X 100 and
188
0.2% BSA in PBS O/N. Left and right trachea sections were stained using the
189
appropriate primary and secondary antibodies O/N at RT (Table 1). Sections were
190
washed thrice in PBS and mounted using a glycerol-based mounting medium containing
191
DAPI (Vector Laboratories). For all antibodies, negative reagent controls were carried
192
out with no staining observed.
193 194
Light microscopy
195
Quantification of αSMA and NG2 positive cells in the trachea and extrapulmonary
196
bronchi was performed using a Leica DM2500 fluorescence microscope (Leica
197
Microsystems, Milton Keynes, UK). Counts were normalized to the length of each
198
tracheobronchal whole mount (including the extrapulmonary bronchi at the distal end of
199
the preparation). For image acquisition from immunostained tracheobronchal whole
200
mounts and lung sections, 1024 x 1024 pixel RGB-color images were obtained using a
9
Johnson et al.
Pericytes in airway remodeling
201
Zeiss LSM 510 inverted confocal microscope with argon, helium-neon and UV lasers
202
(Carl Zeiss AG, Göttingen, Germany) using an EC Plan-Neofluar 40x/1.30 oil objective
203
with the DIC phase contrast technique (10). Using the optimized pinhole size, 0.9 μm
204
thick optical sections were sequentially collected with a step size of 1 μm (lung sections)
205
or 1.5 μm (tracheas). Image analysis was performed using Zeiss AIM 3.2.2 software
206
and Zeiss LSM Image software (Carl Zeiss AG). Three-dimensional visualizations
207
(Videos 1-4) of confocal images were prepared using Volocity software (Perkin-Elmer,
208
Waltham, MA). Morphometric analysis of ASM thickness was performed by a blinded
209
observer on αSMA-stained lung sections and analyzed using ImageJ software (NIH,
210
USA). Morphometric analysis of the number of NG2+ cells in the airway subepithelial
211
region was conducted by a blinded observer on lung sections stained for NG2, SMA
212
and CD31 (to prevent enumeration of pericytes associated with the microvasculature).
213
Five representative images were taken of the large airways (bronchi and bronchioles)
214
from each animal (n=5-10 per group from two independent experiments). Images were
215
taken on a Zeiss LSM 510 inverted confocal microscope and processed using ImageJ.
216
NG2+ cells in the airway subepithelial region were enumerated and counts were
217
normalized to mm of basement membrane.
218 219
Quantitative PCR (qPCR)
220
Whole tissue RNA from lung was isolated with TRIzol reagent (Invitrogen/Life
221
Technologies, Stockholm, Sweden) and QIAGEN RNeasy (Qiagen, Sollentuna,
222
Sweden) according to the manufacturers’ instructions. Total RNA (1 μg) was reverse
223
transcribed according to the manufacturer’s instructions (iScript cDNA synthesis kit, Bio-
10
Johnson et al.
Pericytes in airway remodeling
224
Rad). qPCR was performed using Platinum SYBR green SuperMix (Invitrogen) and
225
25 ng cDNA per reaction. Primers used were: Pdgfb QT00266910, Pdgfrb QT00113148,
226
L19 QT01779218. Ct values for the three genes analyzed were: 19-20 (PDGF-BB); 24-
227
26 (PDGFRβ); 17-18 (L19). Expression levels were normalized to the expression of
228
L19. Melt curves were assessed following the completion of RT-PCR to ensure that the
229
fluorescent signal resulted from a single PCR product.
230 231
Data analysis
232
Data were analyzed with GraphPad Prism Software version 6 (GraphPad Software, San
233
Diego, CA, USA). Statistical analysis was performed by Student’s t-test or one-way
234
analysis of variance (ANOVA) with a subsequent Tukey post-hoc test where
235
appropriate. Differences were considered statistically significant when p
